Substantial progress has been made in developing genes and transformation technologies to reduce disease transmission by genetically modified mosquitoes. The next challenge to implementation of transgenic mosquitoes is development of tools to drive the spread of transgenes from small numbers of laboratory- manipulated mosquitoes into field populations. The intracellular bacterium, Wolbachia pipientis, provides an attractive drive mechanism, because it favors its own transmission through a reproductive distortion called cytoplasmic incompatibility. A major challenge to implementation of Wolbachia arises from its obligate intracellular lifestyle. Systematic manipulation of Wolbachia in cloned mosquito cell lines has not yet been attempted, yet is essential to development of Wolbachia as a tool for driving transgenes into mosquito populations. We propose to define physiological conditions of the mosquito host cell that favor uniform growth and recovery of viable Wolbachia. We will clarify whether an undescribed virus infects the uncloned Aa23 population of mosquito cells, which provides the source of Wolbachia for introduction into other, cloned mosquito cell lines. We will use biochemical approaches to determine whether the pleiomorphic forms of Wolbachia include stages that are particularly amenable to transformation. We will transform Wolbachia to express trimethoprim resistance and green fluorescent protein using homologous and transposon-based approaches. We will manipulate folic acid levels in mosquito cell culture media to require the mosquito cell to maintain Wolbachia as the source of folic acid, an essential vitamin. These efforts take advantage of the metabolic capabilities of Wolbachia predicted from genome sequences. Our goal is to develop a user- friendly, persistently-infected mosquito cell line that maintains uniform levels of Wolbachia. Success will improve our understanding of the growth and development of an intracellular microorganism with important potential for mosquito control, provide a basis for direct manipulation of the Wolbachia genome, and provide new insights relevant to development of novel cytoplasmic incompatibility phenotypes that can drive successive sweeps of transgenes through mosquito vector populations. [unreadable] [unreadable] [unreadable]